Human botulism can result from the ingestion of food contaminated with botulinum neurotoxin (BoNT), from the infection of wounds or intestine, or from direct inhalation of the toxin, which can be transcytosed across epithelial barriers as a physiologically active molecule. Intoxication, if untreated, may lead to death due to respiratory muscle failure, while recovery from botulism is a prolonged process (up to several months) and may require intensive and expensive hospital care. Due to their extreme toxicity and ease of production, BoNTs are classified as one of the six highest-risk threat agents for bioterrorism by the US Center for Disease Control and Prevention (CDC). Developing antidotes to botulinum neurotoxins is a priority goal of the Biodefense Research Agenda. Our laboratory has developed genetic constructs and expression systems based on recombinant, full- length, atoxic BoNT derivatives that preserve key structural features of the wt BoNT/A required for physiological trafficking, and enable the development of novel anti-BoNT therapeutics (ABTs) that will be effective in a post-exposure setting by oral or inhalational routes. The construction and expression of these atoxic BoNT derivatives provide molecular delivery vehicles that target motor neurons, and can contain specific sites for enzymatic attachment of a wide variety of cargo and tracker molecules. The genetic constructs are modular by design, and enable the facile production of diverse candidate ABTs for evaluation. In this application we will test two approaches for delivering therapeutic moieties to target neurons: a) For the ABT-1 approach, therapeutic moieties effective against all BoNT serotypes will be incorporated directly into the amino acid sequence of the BoNT atoxic light chain (LC) metalloprotease, which will be expressed in combination with the BoNT/A heavy chain (HC) as full-length ABT-1; b) For the ABT-2 approach, a therapeutic moiety specifically active against wt BoNT/A will be coupled to the full length ABT-2 by site- selective enzymatic attachment to the ABT-2 precursor, carried by the BoNT/A heavy chain. The full-length BoNT derivatives we propose are expected to have unique advantages with respect to their ability to compete with wild type toxin for neuronal binding sites, and to mimic the endosome-to-cytosol channeling required for delivery of ABTs to the neuronal cytosol. Our current methods can produce sufficient quantities for in vitro and in vivo testing. These ABTs can be administered in a post-exposure setting by oral or inhalational routes.